Telomerase and human papillomavirus (HPV) DNA were evaluated as potential markers of high-grade dysplasia in cervical cytological specimens. Cytology specimens were collected from patients at the time of colposcopic evaluation for management of a previous abnormal cytology test result. Telomerase activity was evaluated by the telomeric repeat amplification protocol (TRAP), and HPV DNA was detected by polymerase chain reaction with L1 consensus-sequence primers and filter hybridization genotyping. Telomerase was detected in 8 of 97 (8.2%) cases with normal cytology or benign cellular changes, in 7 of 98 (7.1%) cases of atypical squamous cells of undetermined significance (ASCUS), in 3 of 95 (3.2%) cases of low-grade squamous intraepithelial lesion (LSIL), and in 17 of 48 (35.4%) cases with high-grade squamous intraepithelial lesion (HSIL). High-risk HPVs were detected in 23 of 97 (23.7%) cases with normal/reactive cellular changes (RCC) cytology, in 28 of 98 (28.6%) cases of ASCUS, in 69 of 95 (72.6%) cases of LSIL, and in 35 of 48 (72.9%) cases of HSIL. Telomerase expression did not correlate with the detection of high-risk HPVs in any cytological diagnostic categories. Telomerase and HPV test results of cytological specimens were correlated with the histological diagnoses of concurrent cervical biopsy specimens. Telomerase showed a sensitivity of 29.9% and a specificity of 94.0% for biopsy-confirmed cervical intraepithelial neoplasia (CIN) II/III. In contrast, high-risk HPVs were detected in 70.1% of cases with underlying CIN II/III, with a specificity of 62.5%. A relatively high proportion of normal/RCC or ASCUS cases with telomerase-positive test results had underlying high-grade dysplasia on cervical biopsy. Thus, technical and practical limitations of the TRAP assay in cervical cytology specimens limit the practical application of telomerase as a diagnostic adjunct in cervical cytopathology.